Parallel process for protein or virus separation from a sample

ABSTRACT

A sample is divided into a series of aliquots with the aliquots being subjected to at least two successive parallel separation steps in order to resolve protein or viral components thereof. The separation steps are performed not only on a sample but subsamples each containing a prelabeled tag to afford comparisons between subsamples. The parallel separation is amenable to high throughput and automation.

RELATED APPLICATION

This application claims priority of U.S. Provisional Patent ApplicationSer. No. 60/481,223 filed Aug. 13, 2003, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates in general to separation methods for aprotein-containing sample for the purpose of identifying or measuringthe sample's constituent proteins, and in particular to the use ofstepwise gradients to identify and/or measure a sample constituentprotein.

BACKGROUND OF THE INVENTION

In life science research it is often desirable to identify theconstituent proteins in a sample. Typically, the sample is extractedfrom an organism or collection of living cells. Such samples, of whichblood serum and cell lysates are representative, are generally composedof many thousands of proteins. In disease or pathway research it isoften necessary to assess the protein composition of many such samplesin order to correlate the presence, absence or amount of specificproteins to the state of the source organism.

Complex mixtures of proteins are typically separated by multiplemechanisms. Common examples of separation parameters are charge,hydrophobic interactions, affinity and molecular weight. Afterseparation into constituent proteins, the identification of constituentproteins is often required. The most common and useful method of proteinidentification is peptide mass fingerprinting using mass spectrometry.FIG. 2 shows an exemplary prior art process flow diagram. This processuses one, two or more methods (61, 62) for separating constituentproteins in the sample mixture, breaking up the proteins in the sample60 into peptides with proteolytic digestion 63, most commonly using thetrypsin enzyme, and reading the mass spectra of the peptides on a massspectrometer 64. Depending on the separation mechanisms used, digestionmay be performed before either one of the two separations or just beforethe mass spectrometry measurement. Also, there may be more or fewerseparation mechanisms than the two shown in the figure. The resultingmass spectra are compared with peptide spectra from theoretical digestsof sequences of known proteins in a database 65 and a plurality ofsample protein identifications 66 are produced by correlation of themeasured peptide masses to the calculated sequence masses.

Several types of mass spectrometer instruments are used for peptide massfingerprinting. One type is the Matrix Assisted Laser DesorptionIonization-Time Of Flight (MALDI-TOF). Peptide samples are introducedinto MALDI instruments by spotting the liquid solution onto a MALDItarget plate, the target plate having been previously coated with amatrix substance that facilitates the ionization of compounds to bemeasured. The MALDI plate with one or more samples spotted upon one ormore of its target areas is then inserted into the spectrometer. A laserbeam ionizes the sample spots and ejects the ions into the drivingelectric fields of the mass spectrometer. An example of a MALDI massspectrometer is the PerkinElmer prOTOF 2000 orthogonal MALDI which uses96-, 384- or 1,536-sample MALDI plates with the form factor of flat,thin microplates.

Another type of mass spectrometer instrument used for peptide massfingerprinting is the electro-spray ionization mass spectrometer (ESI).Sample introduction of ESI instruments may be a continuous ornear-continuous flow of liquid unlike the batch loading of discretesamples required by the MALDI. In this continuous flow case,measurements are taken serially at periodic time intervals against acontinuous inflow of peptides to be characterized.

Separation of the protein mixture may be performed in a variety ofseparation matrices. A separation matrix is a support that has size,porosity and functionality characteristics in order to enableinteraction with, and separation of, molecules. Typical supports forseparation matrices include silica, alumina, agarose, acrylamide,styrene divinylbenzene, glass, dextran, polystyrene, acrylics, nylon,polyvinylidene difluoride, and combinations thereof. The separationmatrix support can be in a form typically found for chromatographyresins such as particles, gels, membranes or any other form that enablessuitable separation characteristics. A flow-through vessel that holds aseparation matrix is commonly called a column.

The functionality characteristics of the separation matrix supportenable interactions with molecules. These functionalities can becationic or anionic to allow for charge based interactions; alkyl chain,usually in the three to eighteen carbon length to allow hydrophobicinteractions; or affinity ligands for specific binding interactions. Thesupport may also have porosity characteristics that cause a molecularweight based separation as the molecules flow through it.

The most established analytical method of separating and identifyingproteins is two-dimensional gel electrophoresis (2-D gel) followed byMALDI mass spectrometry. The major steps of this process are shown inthe flow diagram of prior art FIG. 3. The complex sample mixture 67 isfirst separated by charge (pH) by an electrophoresis process calledisoelectric focusing 68. This produces a linear strip of gel materialwith the proteins separated by charge along the length of the strip. Thestrip is placed in contact with the edge of a two-dimensionalpolyacrylamide gel sheet in the appropriate buffer and voltage isapplied to separate the proteins according to molecular weight via gelelectrophoresis 69. Individual proteins form spots of varying sizes andshapes across the gel. The proteins are labeled 70 either before orafter separation, typically either through staining or fluorescentlabeling. The labeled 2-D gel is imaged and the image is analyzed 71 toidentify specific spots representing the location of specific proteinsin the gel. The spots of proteins of interest are cut out of the gel 72and digested to peptides 73, typically with trypsin. The peptidesolution is then typically spotted onto a MALDI plate to facilitatepeptide mass fingerprinting using a mass spectrometer 74 and a massdatabase 75 to produce protein identifications 76.

A common extension to the 2-D gel process is assessment of differentialprotein expression between two complex samples, samples from normal anddiseased organisms for example. One typical process for differentialseparation on 2-D gels is to label all of the proteins in each samplewith a different fluorescent dye (Patton et al. Current Opinion inBiotechnology 2001 6:63-69). The samples are then mixed, the 2-D gelseparation is performed, and then the imaging is performed separately atthe wavelengths of each of the two fluorescent dyes. In theory, proteinsthat exist in common in both samples will produce 2-D gel spots that arecoincident. Proteins that exist in one sample but not in the other willproduce spots at only one of the wavelengths. Further, proteins thatexist in both samples but in different concentrations can be assessed bythe ratio of their fluorescent intensities at the two wavelengths.Another typical process for differential measurements on 2-D gels isdigital correlation of protein spots in images from two independentgels, and quantitating the differences in protein amount in each gel.This method suffers from its dependence on multiple 2-D gels producingprotein spots in a reproducible manner.

Among the shortcomings of the 2-D gel process are the degree of skillrequired to perform the process, the large amount of manual manipulationof reagents and gels required, the lack of repeatability andreproducibility of results, and the length of time required for theprocess, which is often two or three days. Also, the assessment ofdifferential protein expression using two dyes is limited by the dyes'ability to label all proteins to produce fluorescent signalsproportional to their concentrations and by the fluorescent dyes'effects on the separation process, as well as limits to spot finding andquantitation at the image processing step. In an attempt to addressthese shortcomings another approach to the task called MUlti-DimensionalProtein Identification Technology (MUDpit) has been developed asdepicted in prior art FIG. 4.

The MUDpit process utilizes liquid chromatography (LC) rather than gelelectrophoresis as the separation modality. Referring to prior art FIG.4, a protein sample 80 is first digested to peptides 81, then LC drivesthe peptide sample mixture through a flow-through column 84 containing aseparation matrix while varying the concentration of the separationbuffer, typically with a constant fluid flow rate and a linearconcentration gradient with time. The buffer concentration gradient istypically produced by linearly varying the flow rates of two buffersolutions 82 and 83, one flow rate increasing while the other decreases,keeping the total flow rate through the LC column constant.

Unlike 2-D gels which produce separations as physical spots withspecific locations on a 2-D plane, LC produces a series of volumes ofeluted solutions (fractions) that are typically sampled at uniform timeincrements from a flowing output stream at the output port of a column85. The LC process is inherently serial in nature; the fractions aredelivered out of a single column one after the other. The MUDpit processfurther utilizes two complete LC processes in series to produce twodimensions of separation analogous to the 2-D gel process. The firstseparation is generally performed on an ion-exchange column and thesecond on a reverse-phase column. Time increment fractions are collectedfrom the output stream of the first column 88, then each of thosefractions is run independently on the second separation column 89 togenerate a second series of time-increment fractions 90. Often theoutput of the second column is directed continuously to the input of amass spectrometer, typically an electro-spray tandem mass spectrometer91. In this arrangement the continuous flow from the second column isdirected to the mass spectrometer instrument and the time-incrementfractions are generated by the mass spectrometer's sampling of thestream. Other variations of MUDpit utilize multi-modality columns,capillaries and other variations of detailed configuration but retainthe significant operational details described here.

The MUDpit process can be adapted to differential analysis between twosamples by labeling the proteins or peptides with mass tags prior toseparation (Patton et al., Current Opinion in Biotechnology 200213:321-328). Mass tags are molecules of known, small molecular weightthat can be resolved by the mass spectrometer but do not materiallyaffect the separation process. Mass spectra of identical peptides fromtwo mass tag-labeled samples will have the same form but will be shiftedalong the mass axis by the difference of the mass of the tags, so theirspectra can be differentiated. The ratio of the paired spectra's signallevels are representative of the relative concentrations of the proteinin the two samples.

The use of mass tags for differential protein analysis has beendescribed extensively in the literature. Mass tags can be isotopes ofthe constituent atoms of the proteins, such as N¹⁵, C¹⁴ or H² or can belarger such as a CH₃ group replacing a hydrogen atom. Labeling proteinswith mass tags can be performed biologically in cell culture by using aculture media containing isotopic compounds as has been described by Odaet al., PNAS Jun. 8, 1999; 96(12):6591-6596 and Chen et al., Anal. Chem.Feb. 16, 2000; 72, 1134-1143, for example. Mass tags can also be applieddirectly to proteins by chemical labeling as described by Weckwerth etal. (Rapid Commun. in Mass Spectrom. 14, 1677-1681; 2000) and Kelleheret al. (Journal of Biological Chemistry, Vol. 72, Dec. 19 1997,32215-32220).

An advantage of the MUDpit process over the 2-D gel process is thedegree of automation that can be applied. The LC process is typicallyhands free. The output of the final LC column can be plumbed into anelectro-spray mass spectrometer to deliver the samples to themeasurement instrument automatically.

The MUDpit technique also has disadvantages. First, the proteins must bedigested to peptides before any separation is performed. This limits theresolution and range of separations as it makes the peptide mixture forthe first separation an extremely complex one with potentially millionsof different peptides to be discriminated. Short peptides may evenoverlap between multiple proteins. Second, the dynamic range of the LCprocess on peptides is lower than that of 2-D gels on intact proteins,so the signals from peptides from high-abundance proteins are morelikely to overwhelm signals from low-abundance proteins. These problemsare more pronounced when using MUDpit for differential measurements onlow-abundance proteins. Further, since the separation elements in MUDpitare inherently serial rather than parallel, the throughput of theprocess is limited, making the elapsed time to evaluate a sample longeven though the process can be largely automated.

Thus, there exists a need for an automated method, system, apparatus andkit for separation and identification of proteins that are morereproducible than 2-D gels. Additionally, the ability to avoid proteindigestion prior to the first separation process and allow separations tobe done in parallel would also prove beneficial. Such a method andsystem that supports differential protein analysis when needed wouldalso prove beneficial.

SUMMARY OF THE INVENTION

A process for separating proteins or viruses within a sample includesdividing a sample containing proteinaceous or viral components intomultiple aliquots. The multiple aliquots are applied in parallel to afirst separation step to yield partially resolved eluates. The partiallyresolved eluates are then subjected in parallel to a second separationstep. Subsequent to the first separation step, digestion of thepartially resolved eluates is optionally performed. Analysis offractions derived from the second separation step containing digestedfractions in combination with analysis of undigested materialcorresponding to the same aliquot often facilitates characterization.Prior to separation, a subsample is optionally labeled with a unique tagand combined with another subsample to yield the separation sample. Taganalysis provides information about the relative quantity of aparticular constituent between subsamples after separation according tothe process detailed herein.

A kit is provided that includes two separate separation steps andinstructions for the parallel separation of a proteinaceous or viralcomponent containing sample through the use of separation buffers. A kitand process as detailed herein are particularly well suited to providesamples for subsequent mass spectrometry analysis to generate acharacterization library for constituent components.

Additional efficiencies are achieved through the use of a system forproteinaceous or viral component containing sample separation thatincludes a pipetting robot, a first separation matrix, and a secondseparation matrix, the first separation matrix and second separationmatrix maintaining well addresses therebetween for optimal efficiency.Labeling the first separation matrix and second separation matrix with amachine-readable label combined with a machine reader and gripper robotcoupled to the pipetting robot further enhances automation andefficiency of the system.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a process flow diagram according to the present invention forparallel separation;

FIG. 2 is a prior art process flow diagram of the general method ofprotein separation and identification by peptide mass fingerprinting;

FIG. 3 is a prior art process flow diagram of the prior art method ofseparating and identifying proteins using two-dimensional gelelectrophoresis followed by peptide mass fingerprinting;

FIG. 4 is a prior art process flow diagram of the prior art method ofseparating and identifying proteins using two-dimensional liquidchromatography followed by peptide mass fingerprinting;

FIG. 5 is a schematic of four separation columns being washed by buffersof four different concentrations, c₀ to c₃;

FIG. 6 is a schematic of the four separation columns of FIG. 5 beingeluted into four collection vessels by four concentrations of the samebuffer, c₁ to c₄;

FIG. 7 is a perspective view of a two-dimensional array of separationcolumns in the form of a deep-well 96-well microplate, depicting as aninset a well forming an open-ended vessel with the separation matrixmounted within;

FIG. 8 is an exploded view of the 96-well microplate separation columnassembly of FIG. 7 configured with a vacuum manifold and a deep-wellplate oriented to draw eluent through the separation columns and intothe wells of the collection plate;

FIG. 9 is an exploded view of the 96-well microplate separation columnassembly of FIG. 7 configured with a vacuum manifold and a 96-targetMALDI plate oriented to draw eluent through the separation columns anddirectly onto the target spots of the MALDI plate;

FIG. 10 is a partial process flow diagram of an embodiment of theparallel separation method of the present invention where multipletime-increment fractions of eluent are taken from the second separationcolumns; and

FIG. 11 is a process flow diagram of an embodiment of the parallelseparation method of the present invention where mass spectra of theintact proteins eluted from the first separation columns are measuredwith mass spectrometer in addition to constituent peptides of theproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has utility as a method for parallel automatedprotein or virus separation and identification. The ability to detectand optionally purify proteins or viral populations from a given samplein a parallel manner according to the present invention affordsefficiency and speed compared to conventional sequential techniques. Theparallel analysis of the present invention is amenable to rapid fielddetection of proteinaceous or viral pathogens associated with diseaseoutbreaks, bioweapon screening, and the like. A sample is divided into aseries of aliquots with the aliquots being subjected to at least twosuccessive parallel separation steps in order to resolve proteincomponents or viral components therefrom. A digestion after the firstseparation or second separation often facilitates subsequent analysis.

While the present invention is detailed hereafter with respect toprotein analysis, it is appreciated that the present invention islikewise well suited to analyze a sample containing multiple viralgenera, multiple viral species, or multiple viral strains. Such a viralanalysis is of value in evaluating the therapeutic effects of anantiviral treatment on the incident viral population.

Protein or virus concentrations are readily discerned followingseparation steps as detailed by resort to a conventional correlativeanalysis technique such as mass spectrometry, fluorescence labelingtags, radioactive labeling tags, and binding assays. Differentialconcentrations are obtained for multiple samples through the use ofcorrelative mass tags, tags as detailed above, or binding assays.Subsamples each having a unique tag are readily mixed to form a sample.Label analysis provides comparative data between subsamples.

One aspect of the present invention is the use of multiple separationcolumns in parallel. A general process flow diagram is shown in FIG. 1,where the starting protein sample mixture 1 is divided into napproximately equal aliquots diluted with a buffer 2. The number ofaliquots, n, is both the degree of parallelism in the subsequentseparation step and the number of fractions into which the sample isseparated in a first separation step. These sample aliquots are nextdelivered to each of n first separation columns 3. The first separationstep provides at least partial resolution of proteins or viruses withina sample aliquot of the parallel separation process based on separationtechniques illustratively including separation based on charge,molecular weight, and hydrophobicity. In the preferred embodiment, thefirst separation step is an ion-exchange separation. The Vivascience®plate represents a commercially available matrix well plate suitable forion-exchange separation.

A first set of separation buffers, the members of the set numbering n+1,are used for the first separation step. The buffers in this set areformulated in any number of ways including variation in components,concentration, pH, ionic strength and the like. Preferably, the bufferset varies monotonically with increasing or decreasing steps ofconcentration c₀, c₁, c₂ . . . c_(n) (hereinafter referred to as astepwise gradient) corresponding to the desired range of firstseparation conditions.

In the instance of ion-exchange, the parallel sample aliquots are thenwashed with the set of first step gradient separation buffers as shownat 4 in FIG. 1, where sample aliquot 1 is washed by c₀, sample aliquot 2is washed by c₁, and so on through the n samples. After washing, thesample aliquots are all eluted in parallel with a set of buffers 5.Preferably in the instance of ion-exchange separation, the buffer usedfor elution is a concentration variant. More preferably, the elutionbuffer is shifted by an increment of concentration. With separationtechniques such as size exclusion, elution occurs with the separationbuffer until the sample proteins or viruses travel beyond the separationmedia. In this illustrative example, sample 1 is eluted with c₁, sample2 with c₂, and so on. Each sample fraction in this illustrativeembodiment has unique but separate conditions where the conditions areadjacent in concentration space in a most preferred embodiment.

The outputs of this first separation process 6 are n partially resolvedeluate solution fractions. In the instance of first separation being byion-exchange, the separation is by charge (pH) if the first set ofseparation buffers utilized a stepwise gradient of pH or separated byionic strength if the first set of separation buffers utilized astepwise gradient of salt concentration. These partially resolved eluatefractions 6 are composed of intact proteins or viruses separated by oneparameter.

With further reference to FIG. 1, the protein fractions are digested topeptides in parallel 7 prior to a second separation step. A variety ofprotein digestion techniques conventional to the art are operativeherein illustratively including enzymatic digestion such as withtrypsin, chymotrypsin, pepsin, or elastase; photolysis; acid hydrolysis;and thermolysis. While the resolved fractions 10 exiting the columns 9are readily used as production purified proteins or viruses, it isappreciated that when the sample contains or may contain unknown speciesthat subsequent analysis occurs. In FIG. 1, there are n peptidefractions 8 that are then delivered to n parallel second separationcolumns 9. The second separation step 9 is appreciated to include any ofthe separation techniques of charge, molecular weight and hydrophobicitywith the recognition that duplicate separation techniques between thefirst and the second separation steps is generally an unproductiveredundancy. The second separation step 9 is also appreciated to be apurification process such as desalting, or concentrating; or a furtherfractionation process. In FIG. 1, the second separation step is depictedas a fractionation process. The output resolved fractions 10 are thendelivered to a mass spectrometer 111 for peptide mass fingerprinting 12.

FIGS. 5 and 6 show in greater detail washing for the preferredion-exchange first separation step 4, FIG. 1, and eluting of step 5,FIG. 1, in a first separation process where n≈4. There are fourseparation columns 15, 16, 17 and 18 along with four correspondingcollection vessels 19, 20, 21 and 22. A subset of first separationbuffers 23 with concentrations c₀ through C₃ are shown washing theseparation columns in FIG. 5. In FIG. 6 an incremented subset ofseparation buffers 24 with concentrations c₁ through C₄ is shown elutingthrough the separation columns, delivering the separated fractions aseluents into the collection vessels 19 through 22.

In the preferred embodiment multiple separation matrices are packagedinto a microplate format, such as the Vivascience Vivawell® 96 IEXion-exchange separation plate kit (Vivascience AG, Hannover, Germany) orthe Millipore Multiscreen Separation System reverse-phase separationplate kits (Millipore Corporation, Bedford Mass.). It is appreciatedthat higher density microplates such as 384-well and higher areoperative herein and as beneficial for performing complex or highresolution. Conventional plates are typically provided in a two-plateset including a separation plate containing the separation matrix and apassive collection plate.

It is appreciated the number of wells in the separation plate and thenumber of parallel separations, n, are independent of one another.While, for instance, a 96-well format makes it convenient to performseparations where n=96, if sufficient separation resolution is obtainedwith n of 48, 32, 24, 16, 12 or less, the parallelism of the presentinvention is still advantageous. With smaller values of n, a largernumber of samples can be separated with one plate. On the other hand, ifan application requires such resolution that n=96 is not sufficient, asample is readily divided into aliquots over multiple plates to achievethe desired resolution the separation technique can support.

FIG. 7 shows a separation plate generally at 25 with a detailed view ofa well thereof 26. A separation matrix 27 is mounted in the well 26 suchthat the input aliquot or partially resolved eluate, with or withoutbuffer, can be placed on top of the separation matrix through the openwell top 28. Eluents that pass through the separation matrix 27 flow outof the bottom of the well through outlet port 29. The eluents can becollected from the port 29. Optionally, centrifugal force, vacuum orpressure is applied to induce eluent flow through the matrix 27.Preferably, vacuum is applied to the outlet port 29 to draw an eluentthrough the matrix 27.

FIG. 8 shows the separation plate 25 and a collection plate 30 with avacuum manifold 31 interposed therebetween. The two plates 25 and 30 andthe manifold 31 are assembled in an effectively airtight stack in theorder shown and a vacuum of from 0.01 to 50 torr is drawn on vacuum port32. This vacuum draws eluent out of each of the wells in the separationplate 25, and allows the eluent to pass unimpeded through the vacuummanifold 31 to be deposited in the corresponding well of the collectionplate 30. It is also appreciated that this configuration is operativewith a first, second or subsequent separation step.

The use of separation plates, such as filter plates, along with vacuummanifolds and collection plates is well established in automatedprocesses such as DNA purification. The use of components with outlinedimensions approximating those of SBA standard microplates facilitatesautomation with a variety of optional robots. In the preferredembodiment, the pipetting robot is a PerkinElmer MultiProbe II™ with theGripper Integration Platform (PerkinElmer LAS, Boston, Mass.). Therobot, when present, executes a predefined sequence of actions definedas a protocol under computer control. The robot grips, lifts, relocates,lowers, and releases inventive separation step such as a microplate aswell as pipetting liquid samples. Using such a robot the stacking ofcollection plate, manifold, and separation plate are automaticallyassembled and disassembled, allowing the pipetting robot access to thewells in either plate whenever needed by the preselected protocol.

The process of FIG. 1 is performed in an automatic hands-free manner bya general-purpose pipetting robot equipped with a gripper actuator andprogrammed and equipped with appropriate labware. The preparation of thesets of step-gradient buffers can be done before the analysis processbegins, or each member of the set can be formulated and mixed in theseparation well by pipetting appropriate volumes of the twoconstituents. The sample to be separated is first diluted to the desiredconcentration with the appropriate buffer, then divided into n aliquotsand delivered to the first separation step wells. The set of firststepwise-gradient separation buffers are then pipetted into the wells.Vacuum is drawn on the first separation wells to deliver n firstseparated protein mixtures in parallel to the wells in the collectionplate. Next, the stack of separation plate and vacuum manifold aredisassembled by the gripper, exposing the collection plate with theseparated intact protein fractions.

The pipetting robot is optionally fitted with one or more barcodereaders. Individual microplates and other reagent vessels andconsumables carry labels with unique identifiers encoded into barcodes.The barcodes are read either by passing the consumable item past astationary reader using the gripper or by a moving barcode readerattached to the robot's moving head. The barcode reader is interfaced tothe pipetting robot's control computer. As a single protein analysisproject may utilize dozens of identical-looking plates and otherconsumables, the robust correlation of sample identification to theresulting data is enhanced by automation. Other types ofmachine-readable identifiers, such as RF ID tags, are appreciated tofulfill the same function.

After the first separation step 4, trypsin and the appropriate buffersare added to the wells of the collection plate and allowed to incubateto digest the proteins 7. The collection plate containing the peptidemixtures 8 is then moved to another position on the robot deck by thegripper, and a new stack of second collection plate, vacuum manifold,and second separation plate is assembled 9. The peptide mixtures 8 fromthe first collection plate are pipetted into the second separation plate9, maintaining the same well addresses in each of the two plates. Thepeptide mixtures 8 are then followed into the wells of the secondseparation plate by the set of stepwise gradient second separationbuffers. A vacuum is then applied to the vacuum manifold drawing thepeptide mixtures 8 in parallel through the second separation plate 9into the second collection plate. The stack is once again disassembledto expose the second collection plate. The separated peptides 10 canthen be pipetted from the plate onto a MALDI target 11.

FIG. 9 shows an alternate configuration of a separation plate 25 andvacuum manifold 31 that may be used with a second or final separationstep. In this configuration the vacuum draws eluent from the sampleplate directly onto MALDI targets 26 on a MALDI plate 35. This avoids anadditional pipetting step that would be required if the eluent iscollected into a second collection plate and then pipetted onto theMALDI targets. The MALDI plate in the preferred embodiment is aPerkinElmer 96-target MALDIchip™ (part number N701 0040), although it isappreciated that a variety of MALDI plates are operative herein. TheMALDIchip™ has SBS-standard outline dimensions and is readilymanipulated by plate-gripping robot actuators.

FIG. 10 shows a process flow diagram according to the present inventionwith an additional dimension of protein separation step. The steps 1-8are precedent to the additional temporal separation steps depicted inFIG. 10. Like numerals used in FIG. 10 have the same meaning as thosedetailed with respect to FIG. 1. After first separation and digestion, npeptide mixture fractions 8 are delivered to n second separation columns40. In this case, m time-increment fractions 41 are eluted from each ofthe second separation columns and collected separately. In the preferredembodiment, m is in the range of approximately 2 to 25 and mostpreferably in the range of 2 to 5. The fractions are drawn in parallelfrom multiple plate wells, but serially in time. In an exemplary case ofreverse-phase second separation columns with m=3, three differentcollection plates or three different MALDI plates are each eluted uponone after another, each receiving a different fraction with differenthydrophobicity. This additional separation step adds a true seconddimension to the automated protein separation process while retainingthe high degree of parallelism and automation.

FIG. 11 shows a process flow diagram according to the present inventionwhere intact proteins as well as digested fragments thereof are bothdelivered for analysis. The steps 1-6 are the same as those detailedwith respect to FIG. 1, to yield n fractions of intact proteins 6. Thesubsequent processes of digestion and second separation are depictedcollectively at 50. The digestion and secondary separation steps arethose detailed herein. The fact that intact proteins have been separatedprior to digestion to peptides has value in narrowing a database search.Fractions of the partially resolved eluates 6 are shunted for analysissuch as measurement by a mass spectrometer 51. Measuring the mass of theintact proteins in addition to the peptide fragments derived therefromprovides an additional constraint on the database search, which in turnproduces a higher success rate of valid protein identifications from thepeptide mass measurements.

A suitable mass spectrometer is an orthogonal MALDI-TOF such as thePerkinElmer prOTOF 2000™. The orthogonal instrument geometry decouplesthe ionization ejection velocity from the electric-field inducedtime-of-flight velocity, enabling the orthogonal MALDI to measure theheavy masses of intact proteins much more accurately than conventionalin-line MALDI or electro-spray mass spectrometers.

The inventive process detailed herein is well suited for performingkinetic or sample constituent ratio studies for a sample containingknown proteinaceous and/or viral constituents. The metabolic orphysiological status of a cell culture or organism is regularly definedby the ratio of various proteinaceous substances. Similarly, variationsbetween viral populations within a host organism or culture affordimportant information with regard to sample origin. Through the washingand elution denoted at steps 3 and 4 with respect to FIG. 1 withseparation buffers preselected with respect to protein or viralconstituents of a sample and otherwise continuing an inventive processas described herein with respect to FIGS. 1, 10 or 11 is operative toyield the metabolic status and/or the identity of a sample origin. Theparallel automation of the present invention affords efficiencies thatmake such previously time-consuming studies practical.

By way of example, bacterial spore age, metabolic state, and straininformation illustratively are derived from a spore lysate samplesubjected to parallel separation according to the present invention withseparation buffers selected for the purification of known sporeconstituents. This aspect of the present invention is particularlyuseful in salmonella, clostridium and anthracis outbreaks in forensicand public health investigations.

Patent documents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. These documents and publications are incorporatedherein by reference to the same extent as if each individual document orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A process for analyzing proteins or viruses in a sample comprising:dividing a sample having a protein or virus component into a pluralityof aliquots; applying said plurality of aliquots in parallel to a firstseparation step to yield a plurality of partially resolved eluates; andsubjecting said plurality of partially resolved eluates in parallel to asecond separation step to yield a plurality of resolved fractions. 2.The process of claim 1 further comprising collecting at least one ofsaid plurality of resolved fractions.
 3. The process of claim 2 whereincollection of the at least one of said plurality of resolved fractionsoccurs onto a MALDI target or plate.
 4. The process of claim 1 furthercomprising the step of analyzing at least one of said plurality ofresolved fractions.
 5. The process of claim 4 wherein analysis is bymass spectrometry.
 6. The process of claim 5 wherein said massspectrometry is performed on a MALDI mass spectrometer.
 7. The processof claim 3 wherein said mass spectrometry is performed on an orthogonalMALDI mass spectrometer.
 8. The process of claim 1 wherein at least oneof said first and said second separation steps separate on a basisselected from the group consisting of: charge, molecular weight, andhydrophobicity.
 9. The process of claim 1 wherein at least one of saidfirst and said second separation steps uses a chromatography resin orchromatography membrane.
 10. The process of claim 1 wherein at least oneof said first and said second separation steps comprises a separationbuffer that varies monotonically between individual aliquots orindividual eluates.
 11. The process of claim 1 wherein at least one ofsaid first and said second separation steps comprises a separationmatrix in linear or two-dimensional array.
 12. The process of claim 11wherein said first and said second separation steps occur with matricesmaintaining well addresses in each of the two matrices.
 13. The processof claim 1 wherein at least one of said first or said second separationsteps occurs within a microplate.
 14. The process of claim 1 furthercomprising: digesting said plurality of partially resolved eluates priorto subjecting said plurality of partially resolved eluates in parallelto said second separation step.
 15. A process for analyzing proteins orviruses in a sample comprising: dividing a sample having a protein orvirus component into a plurality of aliquots; applying said plurality ofaliquots in parallel to a first separation step to yield a plurality ofpartially resolved eluates; subjecting said plurality of partiallyresolved eluates in parallel to a second separation step to yield aplurality of resolved fractions; digesting said plurality of partiallyresolved eluates with a proteolytic enzyme to yield a plurality ofdigested eluates; and subjecting said plurality of digested eluates inparallel to a second separation step to yield a plurality of resolvedpeptide fractions.
 16. The process of claim 15 further comprising:collecting at least one of said plurality of resolved fractions.
 17. Theprocess of claim 16 wherein collection of the at least one of saidplurality of resolved fractions occurs onto a MALDI target or plate. 18.The process of claim 15 further comprising analyzing at least one ofsaid plurality of resolved fractions.
 19. The process of claim 18wherein analysis is by mass spectrometry.
 20. (canceled)
 21. The processof claim 19 wherein said mass spectrometry is performed on an orthogonalMALDI mass spectrometer.
 22. The process of claim 15 wherein at leastone of said first and said second separation steps separate on a basisselected from the group consisting of: charge, molecular weight, andhydrophobicity.
 23. (canceled)
 24. The process of claim 15 wherein atleast one of said first and said second separation steps comprises aseparation buffer that varies monotonically between individual aliquotsor individual eluates.
 25. The process of claim 15 wherein at least oneof said first and said second separation steps comprises a separationmatrix in linear or two-dimensional array.
 26. The process of claim 25wherein said first and said second separation steps occur with matricesmaintaining well addresses in each of the two matrices.
 27. The processof claim 15 wherein at least one of said first or said second separationsteps occurs within a microplate.
 28. The process of claim 15 furthercomprising digesting said plurality of partially resolved eluates priorto subjecting said plurality of partially resolved eluates in parallelto said second separation step.
 29. (canceled)
 30. The process of claim18 further comprising analyzing at least one of said plurality ofpartially resolved eluates prior to digestion in concert with thecorresponding resolved fraction.
 31. The process of claim 30 whereinanalysis is by mass spectrometry.
 32. The process of claim 1 wherein thestep of applying said plurality of aliquots in parallel to said firstseparation step is performed by a robot.
 33. The process of claim 1further comprising affixing a machine-readable label to at least onecollection selected from the group consisting of: said plurality ofaliquots, said plurality of partially resolved eluates, and saidplurality of resolved fractions.
 34. The process of claim 1 furthercomprising: labeling a subsample with a unique tag; and combining saidsubsample with a second uniquely labeled subsample or an unlabeledsubsample prior to said plurality of aliquots. 35-42. (canceled)